A novel sulfatase, specific for the 3-0 sulfate ester of methyl-2-deoxy-2-sulfamino-alpha-D-glucopyranoside 3-sulfate, has been purified from human urine and used as a probe to study the structural composition of heparin. Heparin having high affinity for antithrombin, as distinguished from molecular species having little or no such affinity, has been found to contain 3-0 sulfated and 3,6 disulfated glucosamine residues. Nuclear magnetic resonance studies using definitively synthesized glucosamine sulfates as standards have confirmed this finding. The presence of 3-0 sulfated glucosamine residues is an important new element in the accepted structure of heparin; such residues may be essential to the binding of heparin to antithrombin in its role as an inhibitor of the coagulation of blood.